Fig. 2.

The effect of time of pre-electrophoretic heating at 50 °C on non-reducing SDS-PAGE analyses of a mixture of the intact h2E2 anti-cocaine mAb and its F(ab’)₂, and Fc fragments. A 1.5 mm thick, 7% acrylamide gel was loaded with samples containing 2 μg each of intact h2E2 anti-cocaine mAb, F(ab’)₂, and Fc fragments (10 μL total volume) per well, electrophoresed until the tracking dye reached the bottom of the gel, and stained with Coomassie blue. The entire gel is shown. Pre-electrophoretic incubation in SDS-PAGE non-reducing sample buffer was carried out for the indicated times at 50 °C. The sample in the lane labeled “boil” was boiled for 3 min prior to electrophoresis. The electrophoretic migration positions of various unfolded stages of each species are indicated to the right of the gel, and discrete bands identifiable in each sample are labeled by letters between gel lanes (see 0–3 min sample lanes). In the Figure, discrete bands/folded states noted for the F(ab’)₂ fragment are labeled with green lettering; “A”, “B”, and “C”, while the discrete bands/folded states noted for the intact mAb are labeled with red lettering; “A”, “B”, “C”, and “D”. See Fig. 4A for the hypothesized identification of the 3 bands/differentially folded states for the F(ab’)₂ fragment, and Fig. 4B for the hypothesized identification of the 4 bands/differentially folded states for the intact mAb.